Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Expression and roles of p16 INK4a , p21 Cip1 and p27 Kip1 in the response of lung adenocarcinoma cell line models to long-term treatment with Dex. H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days and whole cell lysates were probed by western blot for p16 INK4a , p21 Cip1 and p27 Kip1 with GAPDH as the loading control (Panel A). H1299GR Clone 4 cells were transduced with either non-targeted control shRNA or P21 Cip1 shRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were extracted and probed by western blot for p21 Cip1 , p27 Kip1 and GAPDH (loading control) (Panel B). In parallel, cells treated as described for Panel B, were plated for colony formation in 6-well plates in triplicates without further treatment (Panel C). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel D). In parallel, cells transfected as described for Panel D, and treated with Dex (100 nM) were plated on days 0 and 5 to measure colony formation in 6-well plates in triplicates without further treatment (Panel E) and also harvested on days 0 and 3 of treatment for measuring cell size (Panel F). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and 24 h later they were treated with Dex (100 nM) or vehicle for 3 days and the cells were stained to assess expression of β- galactosidase (blue staining) (Panel G, left); in parallel, the treated cells were lysed and analyzed by western blot for p27 expression (Panel G, right). H1299GR Clone 4 cells were transduced with either non-targeted control vector or pLenti-p27 expression vector and after the indicated number of days, whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel H). In parallel, cells were harvested for measuring the cell size (Panel I) and also plated to measure colony formation in 6-well plates in triplicate (Panel J). Panel C: *P, 0.0004; **P, 0.0006; § P, 0.0008; ‡ P, 0.00015. Panel E: *P, 0.016. Panel J: *P, 0.015; § P, 0.00006; ‡ P, 0.00007.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Western Blot, Control, Transduction, shRNA, Transfection, Staining, Plasmid Preparation

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Mechanism of accumulation of p27 Kip1 in response to long-term Dex treatment in high GR expressing cells. H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or a combination of FOXO3 and FOXO4 siRNAs and treated with Dex (100 nM) for the indicated number of days, when whole cell lysates were probed by western blot for FOXO3, FOXO4, p27 Kip1 and GAPDH (loading control) (Panel A). H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days, when RNA was extracted and relative expression levels of p27 Kip1 mRNA were measured using real time RT-PCR (Panel B). H1299GR Clone 4 cells were pre-treated with vehicle or Dex (100 nM) for 72 h. The cells were then treated with 1ug/ml of actinomycin D and at the time points indicated, RNA was harvested from the cells and p27 Kip1 mRNA was measured by real time RT-PCR to determine p27 Kip1 mRNA turnover rates. The mRNA values are plotted relative to the values at the time of addition of actinomycin D (Panel C). Panel B (H292): *P, 0.01; § P, 0.008. Panel B (A549): *P, 0.02; § P, 0.0001. Panel B (H1299GRα clone2): *P, 0.0001; ‡ P, 0.0007, § P, 0.001. Panel B (H1299GRα clone4): *P, 0.0001; ‡ P, 0.001; § P, 0.0001.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Effect of Dex on mouse tumor xenografts expressing low or high levels of GR. SCID mice bearing xenografts of parental H1299 cells (Panel A) or H1299GR Clone 4 cells (Panel B) were implanted with either placebo or Dex (2.5 mg) slow-release pellets at the time indicated by the arrow. The tumor growth was monitored via caliper measurements and tumor volumes were calculated using the formula noted in the Methods section. At the time of sacrifice, residual H1299GR Clone 4 tumors were harvested from the placebo (Panel C, left) and Dex (Panel C, right) treated mice and stained to ensure GR expression and to quantify p27 Kip1 by immunohistochemistry (Panel D). The scoring criteria based on staining intensity and percent positivity, as outlined in the Methods section. The parental H1299 cell tumor xenografts harvested at the end of placebo or Dex treatments in Panel A were also examined by immunohistochemistry for GR and p27 Kip1 expression (Panel E). Panel B: *P, 0.08; § P, 0.016; ‡ P, 0.0015.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Oncogene

Article Title: Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK.

doi: 10.1038/sj.onc.1207645

Figure Lengend Snippet: Figure 3 Effect on p21 and p27 expression in DLD-1 cells. (a) Cells were treated with 20-mM of 2,3-DCPE and harvested at various times. Cells treated with DMSO were used as a control for each time point. (b) Cells were treated with different concentration of 2,3-DCPE for 24 h. Cells treated with DMSO for 24 h were used as a control

Article Snippet: Rabbit anti-human p27, ERK, phospho-ERK, p38, phospho-p38, JNK and phospho-JNK, and mouse anti-human p21 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Control, Concentration Assay

Journal: OncoTargets and Therapy

Article Title:

Hypermethylation Of ADHFE1 Promotes The Proliferation Of Colorectal Cancer Cell Via Modulating Cell Cycle Progression

doi: 10.2147/ott.s223423

Figure Lengend Snippet: Figure 3 ADHFE1 modulates cell cycle progression. (A) GSEA analyses of the “REACTOME_CELL_CYCLE” and “REACTOME_G1_S_TRANSITION” gene sets in the low versus high expression group of ADHFE1 in CRC. (B and C) Flow-cytometry analyses of the cell cycle progression in the indicated CRC cells. Error bars represent the means ± SD from three independent experiments. *p<0.05, **p<0.01. (D) Western blot analyses of p53, p21, p27 and CyclinD1 in the indicated cells. GAPDH was used as a loading control.

Article Snippet: The PVDF membrane was subsequently blocked in PBST solution containing 5% non-fat milk and incubated at 4°C overnight with specific antibodies anti-ADHFE1 (1:1000 dilution; GeneTex Inc, Texas, St. Antonio, USA), anti-CyclinD1 (1:500 dilution; ProteinTech Group, Chicago, IL, USA), anti-p21 (1:500 dilution; ProteinTech Group, Chicago, IL, USA), anti-p27 (1:1000 dilution; ProteinTech Group, Chicago, IL, USA), anti-p53 (1:2000 dilution; ProteinTech Group, Chicago, IL, USA) and anti-GAPDH (1:5000 dilution; ProteinTech Group, Chicago, IL, USA).

Techniques: Expressing, Flow Cytometry, Western Blot, Control

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression on quiescent prostate cancer cells. (A) Cell surface HER2 expression on PCa cell lines (PC3, DU145, C4-2B, LNCaP) and benign prostate epithelial cells (PNT2) cultured in 10% FBS measured by flow cytometry and mean fluorescence intensity (MFI). Open histograms represent an isotype control antibody. Shaded histograms are a HER2 antibody. The vertical line and arrow represent positive expression as defined by the isotype control antibody. (B) Flow cytometry for surface HER2 and BrdU incorporated into DNA of PC3 cells (C) Histograms showing HER2 cell surface expression on the same cell lines cultured in the presence or absence of FBS. (D) Quantification of HER2 surface expression from panel (B) as defined by mean fluorescence intensity (MFI). (E) Immunofluorescence imaging of PC3 (left) or C4-2B (right) cells cultured in either 10% FBS or serum starved conditions showing endogenous p27 and HER2 expression. HER2 (red), p27 (green) and nuclei (DAPI, blue). Scale bar, 50 μm. (F) Cell surface HER2 as measured by flow cytometry of PCa cells cultured with either 1 µM abemaciclib or 0.01% ethanol vehicle. (G) Western blot for HER2 from indicated cell lines cultured for 2 days with or without FBS. β-actin was a loading control. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Control, Immunofluorescence, Imaging, Western Blot

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression is associated with p27 expression. Left: PC3, Right: C4-2B. Top: Flow cytometry histograms demonstrating cell surface (labeling before fixation) HER2 expression on PC3 and C4-2B cells. Cells were gated by lowest 10% or highest 10% of HER2 levels (HER2 low and HER2 high) populations. Middle: Example flow cytometry histograms of p27 labeling from either the lowest (blue) or highest (red) 10% of HER2 surface expression. Bottom: Quantified p27 expression in the low vs. higher HER2 populationsp27 levels were measured as mean fluorescence intensity (MFI). Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: For p27 labeling,Cells were fixed with cold 70% ethanol and stained with anti-p27 antibody (R&D systems, Cat #: IC2256V) in PBS containing 1% FBS for 30 min at room temperature.

Techniques: Expressing, Flow Cytometry, Labeling, Fluorescence

Journal: Biomedical Optics Express

Article Title: 3D-printed biosensors in biomedical applications exploiting plasmonic phenomena and antibody self-assembled monolayers

doi: 10.1364/boe.516565

Figure Lengend Snippet: Fig. 11. (a) SPR spectra obtained by the 3D-printed biosensor for different values of p27 concentration. (b) Absolute value of the experimental variation in resonance wavelength (black marker), calculated with respect to the resonance wavelength of the blank, with relative error bars. The Langmuir fitting (red line) of the experimental values is also reported.

Article Snippet: Human recombinant p27 protein was furnished by OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Concentration Assay, Marker

Journal: Biomedical Optics Express

Article Title: 3D-printed biosensors in biomedical applications exploiting plasmonic phenomena and antibody self-assembled monolayers

doi: 10.1364/boe.516565

Figure Lengend Snippet: Fig. 12. (a) Normalized transmitted spectra obtained with the analyte (p27) and two interferents (MMP-2 and IL-6); (b) Experimental resonance wavelength variations (calculated with respect to the blank) for the three analyzed solutions.

Article Snippet: Human recombinant p27 protein was furnished by OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: